An intricate B12 (or cobalamin) trafficking pathway weaves through three cellular compartments and exists ulitmately to support the activites of only two known B12-dependent enzymes in humans: cytoplasmic methionine synthase and mitochondrial methylmalonyl-CoA mutase (MCM). Clinical genetics studies on patients with inborn errors of cobalamin metabolism have led in recent years to the discovery of nine genes dedicated to processing, delivery and utilization of the B12 cofactor. Studies in our laboratory are unearthing a medley of novel chemical reactions that tailor dietary B12 into its active cofactor forms and are susceptible to blockade by antivitamin B12 derivatives with chemotherapeutic potential. Our studies are also providing detailed insights into the mechanism of complex bidirectional signaling between a GTPase-powered chaperone and its B12 target, MCM, which ensures the fidelity of the cofactor loading process. In the next cycle, we will address the following specific aims. (i) Elucidate the early pathway comprising CblC, which converts incoming alkyl- and cyano-cobalamins into a common intermediate for subsequent partitioning into the synthesis of the active cofactor forms, and CblD, whose function is unknown but structurally, exhibits molecular mimicry with respect to CblC. (ii) Elucidate the mitochondrial branch of the pathway comprising adenosyltransferase, which synthesizes the active 5-deoxyadenosylcobalamin cofactor and subsequently transfers it directly to MCM in a process that is gated by a G-protein sentry, CblA. In the past cycle, we have focused our studies on the bacterial orthologs of the mitochondrial proteins but with the recent availability of sufficient quantities of the human proteins and their structures, we propose to move away from models to studying the disease-causing targets directly. Our proposed spectroscopic, cell biological and structural approaches will help decipher the chemical and regulatory strategies deployed by the intracellular B12 handlers and illuminate the corruption of these processes by disease-causing mutations. The impact of the proposed studies is both fundamental (i.e. exposing strategies to sequester, process and deliver a rare and reactive cofactor that might be of broader relevance to organic cofactor trafficking, which is poorly understood) and medical (i.e. assessing the feasiblity of a B12 trafficking protein as an antineoplastic target and informing therapeutic options for circumventing metabolic blockades due to disease-causing mutations in B12 trafficking proteins).